In the fields of biomolecules, pharmaceuticals, food compounds, chemicals, bioelectronics and others, a wide and diverse selection of separation materials is used. These materials range from polymeric materials such as those derived from organic monomers as styrene and divinylbenzene or those based on biopolymers such as agarose or cellulose, to inorganic materials such as those based on silica or hydroxyapatite.
The advantages of inorganic materials, such as silica beads, are their mechanical stability and their highly defined pore structure. For example, the pore size of silica material extensively used for separations in numerous industries, is well defined and has a pore size distribution close to the theoretical or perhaps practical attainable limit. It is generally known that the ability of inorganic materials, such as silica, to organize into highly structured assemblies is much more pronounced than in organic materials.
The importance of pore size distribution and its impact on separation efficiency is described in the Van Deemter equation. One component of this equation:Hm=ωdp2.v/Dm relates to the mass transfer effect on efficiency of separation (Hm) to particle size (dp2), flow velocity (v), diffusion coefficient of the analyte in the mobile phase (Dm) and a coefficient related to pore size distribution and shape (ω). This relationship predicts that as ω gets smaller (narrower pore size distribution), Hm gets smaller (i.e. lower plate height which leads to better separation efficiency). It is well known in the separations industry and is particularly important for separations based on molecular mass, i.e. Stokes radius, such as the separation of peptides, proteins and other oligo- or macro-molecules. Thus, pore size distribution is a key parameter, but it is typically only controllable with any certainty by the use of inorganic resin materials, such as porous silica (see FIG. 2A). Particularly in filtration materials the ‘regularity’ of pore size has been reported to be of great importance. Well defined pore shapes and size enables the filtration process to be selective for particular molecules leading to sharp exclusion limits and high resolution.
Pores are classified according to their diameters, where micropores have diameters less than about 2 nm, mesopore have diameters within the range of about 2 nm to about 50 nm and macropores have diameters greater than about 50 nm.
Although porous silica may yield highly defined spherical or approximately spherical beads, a disadvantage of silica based materials is their well-known instability towards alkaline conditions, often applied in the regeneration steps carried out between separation steps. For example, in protein biopharmaceutical purification, C18 silica is typically used in a final ‘polishing’ separation step. After a small number of protein purification cycles, typically 2, a wash with concentrated sodium hydroxide is usually carried out to remove protein residue and other undesirable materials bound to the chromatographic column that may cause fouling. This wash procedure is often required to conform to certain regulatory requirements, e.g. FDA. The lifetime of such silica beads and their prolonged use in such processes is thus limited. Since the silica that has been degraded by this washing process has to be regularly replaced by new silica, this represents a considerable cost factor for the user. An additional limitation is seen where the separation of basic compounds (many pharmaceutical drugs are ‘basic’) on reversed phase silica columns is contemplated since alkaline conditions are required to be non-charged compounds in order for them to interact with the hydrophobic surface.
Silica is also known to expose undesired chemically active sites on its surface. Despite these limitations, silica is a widely used separation material, mainly due to its strong mechanical stability and absence of swelling in solvents. Furthermore, the highly ordered pore structure contributes to high separation efficiencies.
In recent years, polymeric separation materials have replaced silica in more and more purification processes. This is mostly due to the improved stability and extended lifetime of these stationary phases. For example, styrene-divinylbenzene based stationary phases are more and more common because they are far more stable during the regeneration steps than silica stationary phases.
However, a major drawback of such polymer materials is that their pore size properties are a) not well defined and b) not easily controlled in the preparation process. Commercially available polymeric bead materials of this type (e.g. from Rohm & Haas (Netherlands) or PolymerLabs (UK)) display pore sizes that are much less well defined than equivalent separation materials based on inorganic materials, such as silica, and they frequently also contain a portion of small pores which may be disadvantageous for demanding separations (illustrated in FIG. 2B). Furthermore, the polymer bead materials derive their macroporosity from the use of porogens and have cross-linking densities typically around 20%. Due to these relatively low cross-linking levels the polymer beads will possess low mechanically stability and may exhibit variable swelling behaviour depending on the solvent system used.
Typically, porogens are organic solvents, such as toluene or dichloromethane, that control the porosity during the polymerization of monomers.
For example, during the synthesis of cross-linked polystyrene, pores are formed in the polymer network in the presence of a solvent or porogen. A cross-linking density below 20% usually leads to small pore sizes in the lower nm range (2-5 nm). Such pores are fairly uniform but the polymer is more like a gel and exhibits only a limited mechanical stability, resulting in compression and collapse of the material upon pressure. Also, micropores (i.e. pores smaller than 2 nm) present in the polymer may not be desirable for certain applications. Ideally, to obtain pressure-stable materials and materials that are less prone to swelling in certain solvents, the percentage of cross-linking should be increased. However, if the cross-linking density exceeds a certain value, e.g. 20%, the polymer will become inhomogeneous and large so-called macropores will be produced having a typical size range of 20-50 nm. These pores are irregular and may terminate inside the polymer matrix, leading to poor diffusion and flow-through properties. As a general rule, by using porogens to form pores, the small pores may be quite uniform but the remaining pores will tend to have a broad size range, particularly in polymers having large average pore sizes.
Commercial separation materials, such as Amberlite (Rohm and Haas) or PLRP-S media manufactured by Polymer Laboratories (UK) are typical examples of such conventional macro-porous polymers that feature amorphous internal structures characterized by irregular pores. An example of the experimentally measured pore size distribution of such materials is illustrated in FIG. 2B.
To address some of the above issues, Feibush (U.S. Pat. No. 4,933,372) disclose a process, in which the pore properties of highly defined silica particles are imaged in polymeric beads. In this process silica particles were filled with monomers and then polymerized. This could be carried out, for example, in an aqueous suspension system where hydrophobic monomers were dispersed in water along with hydrophobic silica particles. Through thermodynamically driven partitioning, the monomers accumulated inside the silica beads. After polymerization, the silica-polymer composite was then subjected to a harsh fluoride or hydroxide wash to remove the silica backbone. As a result of this process, polymer beads representing a mirror image of the silica beads were produced. These beads corresponded in size to the starting silica particles—the bulk of the polymer existed where the pores were previously present in the silica while the polymer pores corresponded to the dissolved silica walls.
Even though this method of Feibush led to some desirable properties in the resulting polymer beads, the complexity of its production process with the associated poor cost-benefit factors precluded its widespread use. The current cost of such premium silica materials lies in the range of a few thousand ε per kg material. In contrast, the non-porous particles required to prepare the separation material, according to the present invention, costs only a few ε per kg.
In a further development, Mallouk et al (Johnson S A., Ollivier P J. and Mallouk T E., Ordered mesoporous polymers of tunable pore size from colloidal silica templates. Science, 1999, 283, 963-965) used colloidal silica to create porous polymer materials. More precisely, a pellet made from dry colloidal silica was made by means of a pellet press. The pressed pellet was then used as a mould; the pellet was produced under a very high pressure of 10000 kPa and at extremely high temperatures, namely 800° C. The process of producing materials under high pressure and heat is termed sintering. A tabular pellet with the dimensions 0.7 cm in diameter and 0.3 cm thickness was obtained. The sintering step was performed with the aim of creating a network of connected colloidal silica particles. The sintered silica particles formed a three-dimensional, interconnected network of colloidal silica. Into this sintered pellet, a monomer solution was used to fill the void spaces between the silica particles and then polymerized. Filling the voids of this interconnected network with monomers, followed by polymerization and subsequent removal of the colloidal silica yielded a continuous porous system in the final polymer. The publication by Mallouk also shows that the pores obtained have a relatively narrow distribution and correspond to a certain extent to the original colloidal silica. However, the work carried out by Mallouk's group does not disclose a process to produce spherical or approximately spherical polymer material useful for common separation or purification applications. In contrast, it provides a cumbersome method of composite formation that is not amenable to any large-scale or industrial process. The colloidal silica used in this work is obtained by a work-intensive sol-gel process including an emulsion of tetraethyl orthosilicate, and the process requires at least 2 days until the final product is obtained (further details are described in K Osseo-Asare & F J. Arriagada, Colloids Surf. 50, 321, 1990).
Sueoka et al (U.S. Pat. No. 4,279,752) disclose the preparation of a porous membrane in which fine silica particles (size 0.01 μm) are admixed. The membranes consist of polyvinyl alcohol that is extruded through a slid die into a coagulation bath and the obtained membrane is then further processed with a cross-linking treatment in another bath. In a third bath, the silica is extracted and the membrane is then washed. The obtained membranes have uniform pore sizes around 1 μm as opposed to the size of the pore forming silica (0.01 μm). The document indicates that the fine silica particles aggregate during the admixture process. The material disclosed by Sueoka et al is not useful as a chromatographic packing material, i.e. it does not have the form of beads.
The publication by Derylo-Marczewska et al (Langmuir, 2002, 18, 7538-7543) discloses the use of fumed silica for the preparation of melamine-formaldehyde resins. From the synthetic details, it can be concluded that a bulk material in the form of a block is prepared. The material disclosed by Derylo-Marczewska et al will not be useful as a chromatographic packing material, i.e. it does not disclose beads. Even though the pore size distribution displays a main peak, it also displays a large portion of micropores and it contains other population of pore sizes. Consequently the block material disclosed does not have a uniform pore size distribution. The micropores may be disadvantageous in chromatographic separations leading to undesirable chromatographic effects and ill-defined peak shapes such as peak tailing.
Li et al (U.S. Pat. No. 5,288,763) disclose the preparation of porous polymer particles based on a template polymerization technique. As template, a linear polymer, polyacrylic acid (PAA) is used, which is dissolved in a monomer mixture containing an initiator. Furthermore after dissolution of the PAA monomer mixture it is filtered to remove any insoluble matter. Disclosed is a soluble template, namely PAA, used to create a part of the pores. The obtained particles are argued to have a narrow pore size distribution, which is not disclosed in the reference. The obtained pore size is larger than 1 μm and mentioned to be uniform in Table 1 (assessed by SEM observations as shown in FIG. 4-5) but without providing supporting data. Further, the obtained beads display both macroporous and microporous regions in their porosity. As Li et al state in their patent (column 1, line 47) micropores will lead to undesirable chromatographic effects and ill-defined peak shapes such as peak tailing.
The work by Asher and Liu (WO 0000278 A1) discloses a process wherein a colloidal silica is mixed with water soluble monomers and then polymerized between two quartz plates. The resulting material is a flat sheet with a typical thickness of 0.1 mm. It contains both large voids and smaller pores and does not display a defined porosity. Furthermore, beads are not disclosed.
In chromatography there is a need for a packing material having the form of beads and a narrow pore size distribution, without micropores, which material is amenable to large scale production by an economical method.